The present inventors previously disclosed a genetically engineered biological indicator comprising at least one test organism and at least one reporter gene for producing an indicator enzyme, the reporter gene being taken up by the test organism, the test organism comprising bacterial spores; and at least one repressor gene that inhibits expression of the reporter gene until the reporter gene is exposed to at least one inducer; wherein the genetically engineered biological indicator is a component of a self-contained biological indicator, e.g., a two compartment system such as a vial and cap combination, the two compartment system further containing a growth medium for the genetically engineered biological indicator. See, e.g., U.S. Pat. No. 8,372,624.
In the genetically engineered biological indicator system and method disclosed in U.S. Pat. No. 8,372,624, the repressor gene inhibits expression of the reporter gene until the system is exposed to the inducer. Various problems have been encountered in use of this system and method, including discoloration of the indicator due to degradation of the inducer and/or issues relating to the repressor gene which, like the reporter gene, must be taken up by the test organism. The discoloration has been observed, for example, when the system is heated during the sterilization process or in processing to produce the genetically engineered biological indicator system. It is believed that the inducer, e.g., xylose, is degraded by the heating or other sterilization process (e.g., by oxidative sterilization processes, such as vapor hydrogen peroxide, ethylene oxide, etc.) and turns brown or is otherwise discolored. This browning or discoloration results in interference with detecting any change associated with a result showing the failure of the sterilization. This is, understandably, undesirable, since it results in reduced sensitivity of the test. The inducer was previously required to overcome the effects of the repressor gene. By omitting the repressor gene, there is no longer any need to use the inducer, and thus the browning or discoloration problem resulting from the inducer can be avoided. In addition, as will be recognized, the necessity to include the repressor gene in the vector used to induce the test organism to take up the reporter gene only increases the complexity of the overall process used to manufacture the genetically engineered biological indicator system. These problems have remained in need of a solution.